Recent trials of immune checkpoint blocking immunotherapeutics resulted in increased long-term survival in lung cancer patients. These drugs remove the brakes from natural anti-tumor T cell responses, thereby allowing T cells to attack tumors. However, it is unclear why anti-tumor T cells do not kill tumor cells in the absence of therapeutic intervention. Using a genetically-engineered mouse model of lung adenocarcinoma, we found that immune cells are present in highly organized, lymph node (LN)-like organs that are physically associated with tumors. These structures are called tumor-associated tertiary lymphoid structures (TA-TLS), TA-TLS have been described in lung cancer patients, but not in animal tumor models, and consequently, little is known about TA-TLS formation, development and maintenance. We have shown that TA-TLS arise as a consequence of tumor development and perform critical functions of LNs. Moreover, therapeutically depleting regulatory T cells (Tregs) from TA-TLS results in tumor destruction. Thus, a powerful anti-tumor immune response, capable of eliminating advanced tumors, is constitutively stimulated but also suppressed in TA-TLS. Accordingly, therapeutic strategies that develop or expand TA-TLS would likely have strong anti-tumor effects. Thus, in this proposal, we focus on the mechanisms mediating TA-TLS development and maintenance. Regarding development, we found that physical association of TLS with tumors requires T-cell-antigen expression by developing tumors. Therefore, we hypothesize that the anti-tumor T cell response plays an instructive role in TA-TLS development by recruiting immune cells involved in lymphoid tissue development to the tumor site. In Aim 1, we propose a time course analysis to determine when TLS form and identify cell types present during their genesis. Furthermore, we will determine whether tumor-specific CD8 T cell responses are sufficient to drive TA-TLS development by modulating antigen expression by developing tumor cells. Finally, we aim to define the time window during tumor development when T cells act, determining how temporal restriction of T cell function impacts the recruitment of immune cells involved in lymphoid tissue development. Here, we will also determine whether continued antigen expression is required for TA-TLS maintenance. In Aim 2, we will expand upon the signals and cell types maintaining TA-TLS, given their potential as therapeutic targets. Specifically, as dendritic cells (DCs) and lymphotoxin (LT) signals are critical for TLS maintenance under other inflammatory conditions, we will investigate their requirement for TA-TLS maintenance through depletion and blockade. This proposal combines advanced techniques in cancer biology and immunology to interrogate a fundamental tumor-associated immune structure that has only been described in human cancers. This award will help me develop an independent research program, establish preliminary data that are competitive for further funding, and make significant contributions to cancer immunology.